This project is broadly concerned with an elucidation of the structural, physiocochemical, and reactivity properties of the three currently recognized types of active sites, (Fe(S-Cys)4), (Fe2S2(S-Cys)4), and (Fe4S4(S-Cys)4), found in iron-sulfur proteins and enzymes. The basic approach to this problem is by synthesis of lower molecular weight analogues of these sites followed by investigation of the preceding properties and detailed comparisons of related properties (where known) of proteins and enzymes. Substantial progress has already been made under NIH support, resulting in synthesis and detailed characterization of the three structural types of sites in five or the seven physiologically significant oxidation levels. The following research is proposed in the coming grant year: (1) full structural and physicochemical characterization of (Fe4S4(SR)4)3-, analogues of reduced 4-Fe protein sites; (2) synthesis of (Fe2S2(SR)4)3- and (Fe4S4(SR)4)-, the two remaining analogues not yet obtained; (3) further development of the active site core extrusion method for site structure identification using a 19F nmr method in cases where other protein chromophores render ineffective the present method; (4) investigation of the effect of S/Se replacement on key physical properties of tetranuclear protein sites and analogues; (v) development of methods for selective removal of one thiolate ligand of (Fe4S4(SR)4)2-, affording a vacant coordination site potentially capable of activating substrates whose catalytic conversions in iron-sulfur enzymes may occur at a tetranuclear site.